Shi Huafang, Tschudi Christian, Ullu Elisabetta
Department of Internal Medicine, Yale University Medical School, New Haven, CT 06536-0812, USA.
RNA. 2007 Jul;13(7):1132-9. doi: 10.1261/rna.474707. Epub 2007 May 25.
In Trypanosoma brucei, Argonaute1 (TbAGO1) is an essential component of the RNA interference (RNAi) pathway. While characterizing a TbAGO1 conditional knockout cell line, we discovered that, upon blockage of TbAGO1 transcription, the RNAi response to transfected double-stranded RNA was severely inhibited, although there was no change in the TbAGO1 protein level. This observation suggested that steady-state TbAGO1 was not sufficient to fully support the RNAi response to transfected dsRNA and implicated newly synthesized Argonaute in this phenomenon. Indeed, a translational blockade of TbAGO1 mRNA with an antisense morpholino oligonucleotide resulted in inhibition of the RNAi response, even though the steady-state level of TbAGO1 remained unchanged during the time of the assay. Thus, we concluded that in T. brucei, newly synthesized TbAGO1 is required to support an efficient RNAi response. We speculate that newly processed siRNAs may be preferentially loaded onto newly synthesized TbAGO1, and this mechanism may contribute to the homeostasis of the RNAi pathway.
在布氏锥虫中，AGO1（TbAGO1）是RNA干扰（RNAi）途径的一个重要组成部分。在对TbAGO1条件性敲除细胞系进行表征时，我们发现，在阻断TbAGO1转录后，对转染双链RNA的RNAi反应受到严重抑制，尽管TbAGO1蛋白水平没有变化。这一观察结果表明，稳态的TbAGO1不足以完全支持对转染双链RNA的RNAi反应，并表明新合成的AGO蛋白参与了这一现象。事实上，用反义吗啉代寡核苷酸对TbAGO1 mRNA进行翻译阻断会导致RNAi反应受到抑制，尽管在检测期间TbAGO1的稳态水平保持不变。因此，我们得出结论，在布氏锥虫中，需要新合成的TbAGO1来支持有效的RNAi反应。我们推测，新加工的小干扰RNA（siRNA）可能优先加载到新合成的TbAGO1上，这种机制可能有助于RNAi途径的稳态。
